Method for determining the haemoglobin F content of an erythroid cell

ABSTRACT

The invention concerns a method for determining, by flow cytometry, the hemoglobin F (HbF) content of each erythroid cell of a set of erythroid cells. This method applies in particular to determining the HbF content of each red blood cell of a set of red blood cells. The invention also concerns a method for determining the amount of red blood cells transfused into a patient and for monitoring the therapeutic efficacy of a treatment for sickle cell disease or β-thalassemia.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a U.S. National Stage Entry of International Application No. PCT/FR2017/053015, filed Nov. 3, 2017, which claims priority to French Patent Application No. 1660713, filed Nov. 4, 2016.

TECHNICAL FIELD

The invention relates to a novel method for determining, in vitro, the hemoglobin F (HbF) content of each erythroid cell of a set of erythroid cells, preferably of each red blood cell of a set of red blood cells, by flow cytometry. The invention also relates to a novel method for determining, in vitro, the amount of red blood cells transfused into a patient and to a method for monitoring the therapeutic efficacy of a treatment for sickle cell disease or for β-thalassemia.

TECHNOLOGICAL BACKGROUND

Sickle cell disease is a genetic disease of hemoglobin which was historically characterized by the deformation of red blood cells (RBCs) in response to a decrease in oxygen partial pressure. The red blood cells lose their biconcave (discocyte) shape and are in an elongated, pointed or sickle shape, hence the name sickle cell disease.

Sickle cell disease is due to a mutation of a single nucleotide (GAG→GTG) on codon 6 of the β gene located on the short arm of chromosome 11 (11p 15.5). This mutation results in a structural modification of the hemoglobin β chain which consists of the replacement of glutamic acid in position 6 with a valine so as to form HbS. The major sickle cell syndrome comprises 3 genotypes: a homozygous form (SS), and 2 heterozygous forms (Sβ° Thal and SC which combines the βS mutation and a mutation C characterized by the replacement of the glutamic acid in position 6 with a lysine).

The clinical phenotype of sickle cell disease is characterized, on the one hand, by a large interindividual variability and, on the other hand, by a variability between each event for one and the same patient. The main causes of hospitalization are vaso-occlusive crisis (VOC) and acute chest syndrome (ACS) which can occur at any age. ACS can affect up to 40% of sickle cell disease patients and represents one of the main causes of mortality in adults. Dense red blood cells (DRBCs) and irreversibly sickled cells (ISCs) constitute a subpopulation of SS red blood cells which are highly pathogenic due to their high concentration of HbS which is the main determining factor of polymerization. DRBCs have a high tendency to polymerize and are major factors of the physiopathological mechanisms of sickle cell disease. By virtue of their low deformability combined with a considerable increase in viscosity, DRBCs contribute mainly to the occlusion of the capillaries and the post-capillary venules and to the slowing of blood flow, promoting the appearance of vaso-occlusive phenomena.

In 2010, approximately 5 million carriers of the sickle cell disease trait and 312 000 children with sickle cell disease were counted worldwide, with a high density found in African regions. In 2011, the WHO estimated a prevalence of sickle cell disease trait carriers ranging up to 5% of the worldwide population. Sickle cell disease at the current time represents the most widespread genetic disease in France with a frequency ranging up to 1 birth in 700 in mainland France and 1/2300 with the overseas departments (Bulletin Epidémiologique Hebdomadaire [French Weekly Epidemiological Bulletin] Jul. 3, 2012/May 12, 2015). Mortality rates in children under the age of 5 have decreased considerably over the past few decades due to neonatal screening and to the introduction of preventive treatments for the infections which constitute the main causes of mortality. In France, an improvement in the life expectancy of sickle cell disease patients has also been observed over the past 20 years, with a mortality rate reduced to 35% (Bulletin Epidemiologique Hebdomadaire [French Weekly Epidemiological Bulletin] Mar. 10, 2015). However, the lack of early screening and the limited access to quality treatments and care continue to cause the incidences of mortality in children to increase in developing countries.

The observation of an asymptomatic form of sickle cell disease in SS patients has led to various studies for the purpose of determining the protective effect of hemoglobin F (HbF). This is because the clinical expression of sickle cell disease is attenuated in the perinatal period during which HbF represents the predominant form. A benign form which has genetic traits resulting in an overexpression of HbF is also observed in adults. These patients rarely exhibit a severe clinical manifestation and benefit from a better life expectancy. The protective effect of HbF consists mainly of the inhibition of the formation of the deoxy HbS polymer. Since the polymerization depends essentially on the mean corpuscular hemoglobin concentration (MCHC), the reduction in intracellular concentration of HbS constitutes the principal factor improving clinical expression. In vitro studies carried out on hemoglobin solutions have made it possible to determine the mechanisms of polymerization inhibition by the various hemoglobin variants (HbA, HbA₂, HbF, etc.). A more significant inhibitory effect is obtained with HbF. Added to this anti-polymerizing effect is a high oxygen affinity of HbF which provides better protection of the red blood cells. In sickle cell disease patients, HbF is expressed heterogeneously, the protective effect of HbF gives the red blood cells containing a high HbF content a longer lifetime, up to approximately 90 days, while the SS red blood cells which contain little HbF are rapidly eliminated from the general circulation. To date, increasing the HbF thus constitutes one of the main therapeutic objectives in the treatment of sickle cell disease.

Nevertheless, the increase in the mean HbF content is not always associated with a clinical improvement in sickle cell disease patients. Conversely, a clinical improvement may be observed at low mean HbF contents. These observations suggest the existence of a threshold HbF content in each red blood cell, making it possible to inhibit the polymerization of the HbS, and suggest the need to exceed this threshold with, as a result, a more homogeneous distribution of the HbF in the red blood cells, so that each red blood cell benefits from the protective effect of HbF. It has moreover been shown that, in the absence of treatment, the distribution of HbF in the SS red blood cells is heterogeneous. The determination of the HbF content of each red blood cell is thus of essential importance. Over the past few decades, several techniques have been developed in order to determine the HbF content of each red blood cell. These techniques were initially based on chemical methods (Betke K, Marti H R, Schlicht I. Estimation of small percentages of foetal hemoglobin. Nature. 1959; 184(Suppl 24):1877-8), then on immunological methods (Navenot J M, Merghoub T, Ducrocq R, Muller J Y, Krishnamoorthy R, Blanchard D. New method for quantitative determination of fetal hemoglobin-containing red blood cells by flow cytometry: application to sickle-cell disease. Cytometry. 1998; 32(3):186-90), the purpose of which was to detect a specific population of red blood cells containing a high HbF content, called “F cells”. Although widely used in the diagnosis of fetal-maternal incompatibilities and in the understanding of hemoglobinopathies, these methods are not very sensitive and are limited to a qualitative measurement of F cells (red blood cells positive by immunofluorescence microscopy, immunodiffusion or by flow cytometry, regardless of the HbF expression level). Up until then, the study of the HbF content in each red blood cell was based on this approximative definition of F cells.

There is therefore a real need to develop an easily implemented method for accurately determining the HbF content in each red blood cell of a set of red blood cells in order to be able to explain the clinical response variabilities, to anticipate these clinical response variabilities and thus to adjust the treatment. This would also make it possible to develop new therapeutic approaches for sickle cell disease or for β-thalassemia aimed at increasing the HbF content in each red blood cell and not only the mean HbF content in a patient.

SUMMARY OF THE INVENTION

The present invention aims to provide a new method for determining the hemoglobin F (HbF) content of each erythroid cell of a set of erythroid cells, preferably of each red blood cell of a set of red blood cells, that is of use in the diagnosis and the therapeutic monitoring of sickle cell disease or of β-thalassemia.

According to a first aspect, the invention relates to a method for determining, in vitro, the hemoglobin F (HbF) content of each erythroid cell of a set of erythroid cells, comprising the steps of:

-   -   a) isolating erythroid cells from a sample;     -   b) permeabilizing the membrane of the erythroid cells isolated;     -   c) labeling the HbF of the erythroid cells obtained in step b)         with an anti-HbF antibody conjugated to a fluorochrome;     -   d) measuring, by flow cytometry, the fluorescence intensity         (MFI) of each erythroid cell of a set of erythroid cells;     -   e) determining the hemoglobin F content of each erythroid cell         of the set of erythroid cells.

According to a second aspect, the invention relates to a method for determining, in vitro, the amount of red blood cells transfused into a patient, comprising:

-   -   a) obtaining a sample containing red blood cells from a patient;     -   b) determining the hemoglobin F (HbF) content of each red blood         cell of a set of red blood cells of said sample according to the         method of the invention;     -   c) using the results of step b) to determine the amount of red         blood cells transfused into a patient, said transfused red blood         cells having an HbF content substantially equal to zero (=0 pg).

According to a third aspect, the invention relates to an in vitro method for monitoring the therapeutic efficacy of a treatment for sickle cell disease or for β-thalassemia, comprising:

-   -   a) obtaining a sample containing red blood cells from a patient         having undergone a treatment for sickle cell disease;     -   b) determining the hemoglobin F (HbF) content of each red blood         cell of a set of red blood cells of said sample according to the         method of the invention applied to the red blood cells;     -   c) using the results of step b) in the determination of the         therapeutic efficacy of the treatment for sickle cell disease or         for β-thalassemia, in which a therapeutic efficacy is observed         when at least a predetermined percentage of the red blood cells         of the set of red blood cells has an HbF content which exceeds a         reference threshold and/or when a decrease in the amount of red         blood cells having an HbF content <2 pg is observed.

According to a fourth aspect, the invention relates to a method for treating sickle cell disease or β-thalassemia, comprising:

-   -   a) obtaining a sample containing red blood cells from a patient;     -   b) determining the hemoglobin F (HbF) content of each red blood         cell of a set of red blood cells of said sample according to the         method of the invention applied to the red blood cells;     -   c) when at least a predetermined percentage of the red blood         cells of the set of red blood cells has an HbF content which         exceeds a reference threshold, initiating an appropriate         treatment in the patient.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Human hemoglobins are composed of 4 subunits of polypeptide chains which are identical in pairs. The subunits vary according to the types of hemoglobin and 4 normal hemoglobins can be distinguished in human beings:

-   -   embryonic hemoglobins: hemoglobin Gower I (ζ2ε2), hemoglobin         Portland (ζ2γ2) and hemoglobin Gower II (α2ε2);     -   HbF (α2γ2);     -   adult hemoglobin (HbA) (α2β2);     -   hemoglobin A2 (HbA₂) (α2δ2).

The term “HbF” denotes a protein composed of two subunits of α(alpha) chains and two subunits of γ(gamma) chains. HbF is composed of two types of γ chains: Gγ and Aγ which differ in terms of their residue in position 136 corresponding to a glycine for the Gγ chain and an alanine for Aγ. There are two minor forms of HbF, including HbF1 which represents 10% of total hemoglobin in the fetus. It contains a γ^(X) subunit, the N-terminal end of which is substituted with an acetyl group. Other forms of HbF can be observed but in very small amount.

For the purposes of the invention, the term “content” should be understood in its main sense, that is to say the amount of the entity of interest. The content can be expressed as weight content, for example in picograms (pg).

For the purposes of the invention, the expression “HbF content of each erythroid cell” denotes the amount of HbF in each erythroid cell, taken individually. Preferably, the content (or amount) is expressed by weight, advantageously in picograms (pg). For example, the determination of the HbF content of each erythroid cell of a set of 10 erythroid cells corresponds to the obtaining of 10 values of amount of HbF, corresponding to the HbF content of each of the 10 erthyroid cells taken individually.

For the purposes of the invention, the term “erythoid cell” or “erythroid cells” denotes cells which express hemoglobin, for which it is desired to measure the presence of fetal hemoglobin (HbF) among a set of said cells. For the purposes of the invention, the “erythroid cells” denote the erythroid cells which differentiate at the late erythropoiesis stage, for example the erythroblasts, the reticulocytes or the red blood cells. Said erythropoiesis cells are cells that may be non-circulating, that is to say derived from the bone marrow (for example the erythroblasts) or circulating cells, that is to say blood cells (for example the reticulocytes or the red blood cells) or cells derived from erythroid cells, for example recombinant erythroid cells. For the purposes of the invention, the erythroid cells are capable of containing HbF. In order to avoid any ambiguity, throughout the present description, the terms “erythroid cell” and “erythroid cells” can be respectively replaced, for example, with “red blood cell” or “red blood cells”. For example, the invention relates to a method for determining, in vitro, the hemoglobin F (HbF) content of each red blood cell of a set of red blood cells, comprising the steps of:

-   -   a) isolating red blood cells from a sample;     -   b) permeabilizing the membrane of the red blood cells isolated;     -   c) labeling the HbF of the red blood cells obtained in step b)         with an anti-HbF antibody conjugated to a fluorochrome;     -   d) measuring, by flow cytometry, the fluorescence intensity         (MFI) of each red blood cell of a set of red blood cells;     -   e) determining the hemoglobin F content of each red blood cell         of the set of red blood cells.

For the purposes of the invention, the expression “set of erythroid cells” corresponds to a set of erythroid cells contained in a sample of erythroid cells. It is possible for the set of erythroid cells to range up to 100% of the erythroid cells present in the sample, that is to say all of the erythroid cells present in the sample. Thus, the set of erythroid cells corresponds to between 100% and 0%, 0% being exclusive, of the erythroid cells present in the sample. In one particular embodiment, the set of erythroid cells comprises at least 10 000 erythroid cells, preferably at least 100 000 erythroid cells, preferably between 50 000 and 100 000 erythroid cells. In this particular embodiment, the hemoglobin F content is determined for each of the erythroid cells of a set of at least 10 000 erythroid cells, preferably of at least 100 000 erythroid cells, preferably between 50 000 and 100 000 erythroid cells.

For the purposes of the invention, the term “flow cytometry” denotes a technique well known to those skilled in the art which makes it possible to pass particles, molecules or cells at high speed through a laser beam, while counting them and characterizing them. It is the light re-emitted by the particles, molecules or cells (by scattering or fluorescence) which makes it possible to characterize them according to the desired criterion or criteria. Generally, the particles, molecules or cells are labeled with a fluorochrome which absorbs the energy from the laser and which re-emits the absorbed energy in the form of photons of a higher wavelength. In the context of the invention, the re-emitted light is obtained by specific labeling of the HbF present in the erythroid cells with an anti-HbF antibody conjugated to a fluorochrome.

For the purposes of the invention, the term “fluorochrome” (or “fluorophore”) denotes a chemical substance capable of emitting fluorescent light after excitation by a laser. In the context of the invention, the fluorochrome is coupled to an anti-HbF antibody. Those skilled in the art have available a wide choice of fluorochromes suitable for flow cytometry. All the fluorochromes which can be coupled to an antibody may be used in the context of the present invention. In one particular embodiment, the fluorochrome is fluorescein isothiocyanate (FITC) or phycoerythrin (PE), or a derivative thereof, preferably PE. PE, by virtue of its conformational (steric hindrance) properties has the advantage of having a PE:antibody ratio close to 1. The antibody coupling chemistries are well known to those skilled in the art and the present invention is not limited to a particular coupling chemistry. As it is used here, the term “antibody” refers to immunoglobulin molecules or other molecules which comprise at least one antigen-binding domain. It encompasses in particular whole antibodies, fragments of antibodies comprising an antigen-binding domain (e.g. Fab, Fab′ and F(ab)2, scFv, fragments comprising either a VL domain or a VH domain), monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, primatized antibodies, monospecific antibodies, multispecific antibodies, single-chain antibodies (e.g. of camelid type). The antibodies according to the invention may be antibodies of any type, for example IgG, IgE, IgM, IgD, IgA and IgY, of any class, for example IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2 or of any subclass. In the context of the present invention, the antibody is an anti-HbF antibody, preferably an anti-HbF antibody directed against the gamma chain of HbF. In one particular embodiment, the antibody is a monoclonal antibody.

Generally, monoclonal antibodies can be obtained by the hybridoma technique, the general principle of which is recalled below. Firstly, an animal, generally a mouse, is immunized with the antigen of interest (for example human HbF) or a fragment of the antigen of interest (for example the gamma chain of human HbF), the B lymphocytes of said animal then being capable of producing antibodies against said antigen. These antibody-producing lymphocytes are then fused with “immortal” myeloma cells (for example murine) in order to give hybridomas. Each hybridoma is multiplied in clone form, each one leading to the production of a monoclonal antibody, the properties of recognition of which, with regard to said antigen of interest, may be tested for example by ELISA, by immunoblotting (Western blot), either one-dimensional or two-dimensional, with immunofluorescence, or using a biosensor. The monoclonal antibodies selected in this way are subsequently purified, especially using the chromatography technique. The monoclonal antibodies may also be recombinant antibodies obtained by genetic engineering by techniques well known to those skilled in the art.

For the purposes of the invention, the term “diagnosis” denotes determining a probability or a possibility of having sickle cell disease in a patient.

For the purposes of the invention, the term “sample” denotes a sample containing erythroid cells, said sample having been obtained from a patient, and from which sample it is possible to carry out the invention. Advantageously, the sample is a blood sample, preferably a human blood sample. The sample may be taken from a patient at any time, for example by taking a blood sample.

For the purposes of the invention, the term “homogeneous patient” denotes a patient who has a homogeneous HbF content over all of his or her erythroid cells. The HbF content over all of the erythroid cells thus follows a normal logarithmic distribution. A homogeneous patient may be a patient presenting a hereditary persistence of HbF, β-thalassemia or β-δ-thalassemia. Thus, the term “homogeneous patients” denotes a set of patients each having a homogeneous HBf content over all of their erythroid cells, it being possible for the HbF content to be different from one patient to the other.

For the purposes of the invention, the term “homogeneous sample” denotes a sample of erythroid cells that has been obtained from a homogeneous patient. Thus, the HbF contents of the erythroid cells of a homogeneous sample exhibit a low variance. That is to say that the HbF contents of each of the erythroid cells of the homogeneous sample are concentrated around the HbF content of the homogeneous sample. Advantageously, a low variance is a variance of less than 15%, advantageously less than 10%, less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%.

Method for Determining the HbF Content of an Erythroid Cell

The inventors have demonstrated that flow cytometry is a particularly suitable technique for easily and accurately determining the HbF content of each erythroid cell of a set of erythroid cells.

Thus, the invention relates to a method for determining, in vitro, the hemoglobin F (HbF) content of each erythroid cell of a set of erythroid cells, comprising the steps of:

-   -   a) isolating erythroid cells from a sample;     -   b) permeabilizing the membrane of the erythroid cells isolated;     -   c) labeling the HbF of the erythroid cells obtained in step b)         with an anti-HbF antibody conjugated to a fluorochrome;     -   d) measuring, by flow cytometry, the fluorescence intensity         (MFI) of each erythroid cell of a set of erythroid cells;     -   e) determining the hemoglobin F content of each erythroid cell         of the set of erythroid cells.

Step a) consists in isolating erythroid cells from a sample. This step consists in purifying (or increasing the level of purity) of the erythroid cells from a sample comprising erythroid cells. This step makes it possible in particular to eliminate certain elements (e.g. platelets and white blood cells) that may be present in the sample. Advantageously, the sample is a blood sample, preferably a human blood sample. Numerous techniques are at the disposal of those skilled in the art for isolating erythroid cells from a sample containing erythroid cells. These techniques are generally simple to carry out and generally provide no particular difficulty to those skilled in the art who will know how to adapt them for their use in the present invention. Mention may for example be made of centrifugation, chromatography techniques, or density gradient fractionation. For example, (i) a blood sample is centrifuged, (ii) the pellet (containing the erythroid cells) is collected and (iii) a suitable isotonic buffer (for example a phosphate buffer) is added to the pellet so as to obtain a suspension of erythroid cells, it being possible for steps (i) to (iii) to be repeated one or more times in order to obtain the isolated erythroid cells.

Step b) consists in permeabilizing the membrane of the isolated erythroid cells. This step consists in rendering the membrane of the erythroid cells sufficiently permeable so that the anti-HbF antibody can penetrate into the erythroid cells, while at the same time preserving the integrity of the erythroid cells (without red blood cell lysis). Numerous techniques are at the disposal of those skilled in the art for permeabilizing the membrane of the erythroid cells. These techniques are generally simple to carry out and generally provide no particular difficulty to those skilled in the art who will know how to adapt them for their use in the present invention. Mention may for example be made of the use of a chemical agent, such as a detergent and/or a surfactant (e.g. saponin, SDS and/or Triton). In one particular embodiment, the membrane of the isolated erythroid cells is permeabilized with sodium dodecyl sulfate (SDS). SDS, also known as sodium lauryl sulfate (SLS), is a detergent and surfactant which is strong and ionic, and commonly used. Generally, the sodium dodecyl sulfate is simply added to the erythroid cell suspension.

Generally, in one particular embodiment, the membrane of the isolated erythroid cells is fixed before the permeabilization step. The fixing of the erythroid cell membrane makes it possible to prevent (or to limit) the lysis of the erythroid cells during the permeabilization step. Several chemical compounds are at the disposal of those skilled in the art for fixing the membrane of the erythroid cells. In one particular embodiment, the membrane of the isolated erythroid cells is fixed with sodium azide and/or formaldehyde. Generally, the sodium azide and/or formaldehyde are simply added to a pellet of erythroid cells or to a suspension of erythroid cells.

Step c) consists in labeling the HbF of the erythroid cells obtained in step b) (that is to say the isolated erythroid cells of which the membrane is permeabilized) with an anti-HbF antibody conjugated to a fluorochrome (also hereinafter referred to as “anti-HbF antibody”). Advantageously, step c) is carried out under conditions which allow intracellular binding between the anti-HbF antibody and the HbF. This step makes it possible to obtain “labeled erythroid cells”. Preferably, a sufficient amount of anti-HbF antibody to be able to label all of the HbF is used, for example an excess of anti-HbF antibody is used. In particular, the step c) consists in labeling the HbF of the erythroid cells obtained in step b) with an anti-HbF antibody conjugated to a fluorochrome under conditions which allow intracellular binding between the anti-HbF antibody and the HbF. That is to say that the binding between the anti-HbF antibody and the HbF takes place inside the erythroid cell. Generally, the labeling consists in incubating the erythroid cells, the membrane of which was permeabilized, with the anti-HbF antibody for a period of time sufficient to allow intracellular binding between the anti-HF antibody and the HbF. The period of time is generally a few minutes, for example between 5 min and 20 min.

In one particular embodiment, the erythroid cells are washed after the labeling step. The washing makes it possible to eliminate the anti-HbF antibodies not bound to the HbF. The washing can be carried out with a suitable buffer, for example a PBS buffer. The washing also makes it possible to eliminate the chemical agent that was used to permeabilize the membrane of the red blood cells. The red blood cells thus washed are then used in the flow cytometry measuring step.

Step d) consists in measuring, by flow cytometry, the fluorescence intensity (MFI) of each erythroid cell of a set of erythroid cells. Advantageously, the fluorescence intensity (MFI) of each erythroid cell of the set of erythroid cells is measured independently of the fluorescence intensity (MFI) of the other erythroid cells of the set of erythroid cells. In this step, a set of labeled erythroid cells, that is to say all or some of the labeled erythroid cells, is thus analyzed by flow cytometry. Thus, the fluorescence intensity (MFI) of each erythroid cell of the set of erythroid cells is measured independently of the fluorescence intensity (MFI) of the other erythroid cells of the set of erythroid cells. The fluorescence intensity of each of the erythroid cells taken independently is thus measured in this step. In one particular embodiment, the fluorescence intensity (MFI) is measured for each erythroid cell of a set of at least 10 000 erythroid cells, preferably of at least 100 000 erythroid cells, preferably between 50 000 and 100 000 erythroid cells.

Step e) consists in determining the hemoglobin F content of each red blood cell of the set of red blood cells. The HbF content is determined from the fluorescence intensity measured by flow cytometry. Advantageously, the HbF content of each erythroid cell of the set of erythroid cells is determined independently of the HbF content of the other erythroid cells of the set of erythroid cells. Thus, the HbF content of each red blood cell of the set of red blood cells is determined independently of the HbF content of the other red blood cells of the set of red blood cells. The determination of the HbF content of each red blood cell of the set of red blood cells is carried out by comparing the fluorescence intensity of each red blood cell with a standard curve. The standard curve makes it possible to associate the light intensity measured for a red blood cell with an HbF content.

For example, the standard curve can be obtained in the following way:

-   -   a calibration straight line which makes it possible to correlate         a fluorescence intensity with a fluorophore number is         established. For example, beads conjugated with a known amount         of fluorophore (calibrated beads) can be used. This calibration         straight line makes it possible to determine the amount of         fluorophore per erythroid cell (hereinafter         fluorophore/erythroid cell). Thus, by relating the measurement         of the fluorescence intensity of each erythroid cell to the         calibration straight line, it is then possible to deduce the         amount of fluorophore of each erythroid cell. The         fluorophore:antibody ratio (which may be 1:1), may also be used         to deduce the number of antigens of each erythroid cell, that is         to say the number of HbF of each erythroid cell;     -   the mean HbF content per erythroid cell (MCHbFCo) of a blood         sample obtained from patients having an HbF content that is         perfectly homogeneous over all of their erythroid cells         (“homogeneous patients”) is determined. The HbF content over all         of these erythroid cells follows more precisely a normal         logarithmic distribution. These samples are hereinafter referred         to as “homogeneous samples”. When the erythroid cells are red         blood cells, the homogeneous patients are for example patients         exhibiting a hereditary persistence of HbF or β-thalassemia or         β-δ-thalassemia. For example, it is possible:         -   to determine the mean HbF percentage (that is to say the HbF             proportion relative to the entirety of all the hemoglobins,             expressed as %) of several homogeneous samples having an HbF             content per erythroid cell which is entirely different from             one sample to the other, for example by high-performance             liquid chromatography (HPLC). This mean HbF percentage can             for example be calculated from HPLC chromatograms by             applying the formula: % HbF=(AUC HbF/AUC total Hb)×100 where             % HbF represents the mean HbF percentage over all of the             erythroid cells,         -   AUC HbF represents the area under the curve of the HbF on             the chromatogram,         -   AUC total represents the sum of the areas under the curves             of each type of hemoglobin;         -   to determine the mean hemoglobin content (that is to say all             the hemoglobins) per erythroid cell (MCHCo) of the             homogeneous samples. For this, the concentration of             hemoglobin expressed per liter of blood (for example             obtained with a blood hemogram) is divided by the number of             erythroid cells per liter of blood (for example obtained by             counting the erythroid cells);         -   to determine the mean HbF content per erythroid cell of a             homogeneous sample according to the following formula:             MCHbFCo=(% HbF*MCHCo)/100;     -   the standard curve is established by associating MCHbFCo and a         measurement of the calibrated fluorescence intensity of         fluorophore/erythroid cell.

Advantageously, the comparison of the fluorescence intensity of each erythroid cell, standardized relative to the amount of fluorophores, with a standard curve (i.e. obtained from homogeneous samples), makes it possible to determine the HbF content of each erythroid cell in an automated manner. To do this, use is made of the formula given by the linear regression associating the amount of fluorophores per erythroid cell and the MCHbFCo of the homogeneous patients.

For example, the automation of the comparison of the fluorescence intensity of each red blood cell with a standard curve may be carried out by means of a computer system processor. This computer system may comprise computer memory means in which are stored instructions which, when they are executed by the processor of the computer system, will perform the comparison of the fluorescence intensity of each red blood cell with a standard curve.

Generally, those skilled in the art know how to adjust the parameters of the flow cytometry with the data from the standard curve so as to directly obtain the HbF content of each erythroid cell of the set of erythroid cells analyzed. The flow cytometry will thus be able to automatically categorize the erythroid cells as a function of their HbF content.

Determination of the Amount of Red Blood Cells Transfused into a Patient

The invention also relates to a method for determining, in vitro, the amount of red blood cells transfused into a patient, comprising:

-   -   a) obtaining a sample containing red blood cells from a patient;     -   b) determining the hemoglobin F (HbF) content of each red blood         cell of a set of red blood cells of said sample according to the         method of the invention applied to the red blood cells;     -   c) using the results of step b) to determine the amount of red         blood cells transfused into a patient, said transfused red blood         cells having an HbF content substantially equal to zero (=0 pg).

According to this method of the invention, the red blood cells can be categorized into two very distinct categories. A category of red blood cells containing variable amounts of HbF (corresponding to the red blood cells of the patient, amount >0 pg) and a category of red blood cells not containing HbF (corresponding to the transfused red blood cells_amount=0 pg).

In one particular embodiment, the patient is suffering from sickle cell disease or 3-thalassemia.

Advantageously, the amount of transfused red blood cells makes it possible to determine a percentage of transfused red blood cells relative to the total red blood cells of the patient. This makes it possible in particular to monitor the change in the amount of transfused red blood cells (or in the percentage of transfused red blood cells) and thus to assist in the diagnosis in certain particular cases, for example the distinction between allo antibodies and auto antibodies directed against red blood cells, which may be transfused or the patient's red blood cells.

Monitoring of the Therapeutic Efficacy for Sickle Cell Disease or for β-Thalassemia

The invention also relates to an in vitro method for monitoring the therapeutic efficacy of a treatment for sickle cell disease or for β-thalassemia, comprising:

-   -   a) obtaining a sample containing red blood cells from a patient         having undergone a treatment for sickle cell disease or for         β-thalassemia;     -   b) determining the hemoglobin F (HbF) content of each red blood         cell of a set of red blood cells of said sample according to the         method of the invention applied to the red blood cells;     -   c) using the results of step b) in the determination of the         therapeutic efficacy of the treatment for sickle cell disease or         for β-thalassemia, in which a therapeutic efficacy is observed         when at least a predetermined percentage of the red blood cells         of the set of red blood cells has an HbF content which exceeds a         reference threshold and/or when a decrease in the amount of red         blood cells having an HbF content <2 pg is observed.

Thus, it is possible to monitor the progression in the therapeutic efficacy of a treatment, aimed at increasing HbF, for sickle cell disease or for β-thalassemia over time, before, during or after the treatment of the patient. It is thus possible to monitor the progression of the therapeutic efficacy of known treatments, or of new treatments. It is thus possible to identify new treatments for sickle cell disease or for β-thalassemia which make it possible to increase HbF synthesis in red blood cells.

In one particular embodiment, the reference threshold is 2 pg or more, for example 3 pg, for example 4 pg, for example 5 pg, for example 6 pg, for example 7 pg, for example 7.5 pg (that is to say 7.5 pg of HbF in the red blood cell).

Method for Treating Sickle Cell Disease

The invention also relates to a method for treating sickle cell disease or β-thalassemia, comprising:

-   -   a) obtaining a sample containing red blood cells from a patient;     -   b) determining the hemoglobin F (HbF) content of each red blood         cell of a set of red blood cells of said sample according to the         method of the invention applied to the red blood cells;     -   c) when at least a predetermined percentage of the red blood         cells of the set of red blood cells has an HbF content which         exceeds the reference threshold, initiating an appropriate         treatment in the patient.

For example, the need to treat a patient or not treat said patient can be evaluated. It is also possible to choose the treatment that will be most suitable for the patient. It is thus possible to be sure that the patient receives the most suitable treatment.

In one particular embodiment, at least 50% of the red blood cells of the set of red blood cells have an HbF content which exceeds the reference threshold.

In one particular embodiment, the reference threshold is 2 pg or more, for example 3 pg, for example 4 pg, for example 5 pg, for example 6 pg, for example 7 pg, for example 7.5 pg (that is to say 7.5 pg of HbF in the red blood cell). The article by Steinberg et al. (Blood 2014, 123, 481-485) gives information on the detectable HbF thresholds.

In one particular embodiment, the suitable treatment is hydroxyurea and/or erythropoietin.

FIGURE LEGENDS

FIG. 1A: Cytogram showing the red blood cell populations. Along the abscissa: Log FSC (Forward Scatter) indicating the morphology of the cells. Along the ordinate: Log SSC (Side Scatter) indicating the granulosity of the cells. The selection of the blood cells was carried out on a logarithmic scale for the gains of the FSC and SSC parameters and on a negative control sample.

FIG. 1B: Cytogram showing the histogram of red blood cell fluorescence distribution. Along the abscissa: Log PE-A indicating the fluorescence intensity for the Phycoerythrin fluorochrome.

The signal measured is the area under the curve (A for area) of the variations in fluorescence intensities of each cell. Along the ordinate: red blood cell count.

FIG. 2A: Cytogram showing the red blood cell populations. Along the abscissa: Log FSC-H (Forward Scatter-height). Along the ordinate: Log FSC-W (Forward Scatter-width).

FIG. 2B: Cytogram showing the red blood cell populations. Along the abscissa: Log SSC-H (Side Scatter-height). Along the ordinate: Log SSC-W (Side Scatter-width).

The selection of the red blood cell singlets was carried out on the height and width parameters of the FSC signal first, then on the height and width parameters of the SSC signal.

FIG. 3A: Cytogram showing the histograms of bead fluorescence distribution. Low: histogram of fluorescence distribution of the beads coupled with the lowest fluorochrome (PE) number. Med-Low: histogram of fluorescence distribution of the beads coupled with a medium-low fluorochrome (PE) number. Med-High: histogram of fluorescence distribution of the beads coupled with a medium-high fluorochrome (PE) number. High: histogram of fluorescence distribution of the beads coupled with a high fluorochrome (PE) number. Along the abscissa: Log FL-2 or PE-A indicating the fluorescence intensity for the Phycoerythrin fluorochrome. Along the ordinate: red blood cell count.

FIG. 3B: Linear regression straight line associating the fluorescence of the 4 populations of beads (Low, Med-Low, Med-High and High) and the fluorochrome (PE) number per bead. Along the abscissa: Log of the fluorochrome (PE) number per bead. Along the ordinate: Log bead fluorescence.

FIG. 4A: Cytograms showing the histogram of fluorescence distribution of the red blood cells for the negative control sample, the isotype control sample. Along the abscissa: Log PE-A indicating the flourescence intensity for the Phycoerythrin fluorochrome. Along the ordinate: red blood cell count.

FIG. 4B: Cytogram showing the histogram of fluorescence distribution of the red blood cells for the isotype control sample. Along the abscissa: Log PE-A indicating the fluorescence intensity for the Phycoerythrin fluorochrome. Along the ordinate: red blood cell count.

FIGS. 4C, 4D, 4E, 4F, 46, 4H, 4I, 4J, 4K, 4L, 4M, and FIG. 4N: Cytograms showing the histogram of fluorescence distribution of the red blood cells from samples of patients 1 to 12 respectively. Along the abscissa: Log PE-A indicating the fluorescence intensity for the Phycoerythrin fluorochrome. Along the ordinate: red blood cell count.

FIG. 5: Linear regression associating the HbF content (pg) and antigen amounts per red blood cell (AG/RBC). Along the abscissa: antigen amounts per red blood cell (AG/RBC). Along the ordinate: HbF content (pg).

FIGS. 6A, 6B, 6C, and 6D: Analysis of HbF distribution in patient A and C, respectively, before (D0) and during the treatment with hydroxyurea (M1, M4, M6). Along the abscissa: Log PE-A indicating the fluorescence intensity of the red blood cells for the Phycoerythrin fluorochrome. Along the ordinate: red blood cell count. The red blood cell samples were treated according to the same procedures as those carried out for the red blood cells of the patients serving as the HbF content range.

FIGS. 6E, 6F, and 6G: Analysis of HbF distribution in patient B before (D0) and during the treatment with hydroxyurea (M4, M9). Alond the abscissa: Log PE-A indicating the fluorescence intensity of the red blood cells for the Phycoerythrin fluorochrome. Along the ordinate: red blood cell count. The red blood cell samples were treated according to the same procedures as those carried out for the red blood cells of the patients serving as the HbF content range.

FIGS. 6H, 6I, 6J, and 6K: Analysis of HbF distribution in patient C before (D0) and during the treatment with hydroxyurea (M3, M5, M6). Along the abscissa: Log PE-A indicating the fluorescence intensity of the red blood cells for the Phycoerythrin fluorochrome. Along the ordinate: red blood cell count. The red blood cell samples were treated according to the same procedures as those carried out for the red blood cells of the patients serving as the HbF content range.

EXAMPLES Example 1: Development of a Method for Determining the HbF Content of Each Red Blood Cell: Construction of a Standard Curve

A group of patients having a perfectly homogeneous distribution of the HbF content of each red blood cell was selected from the SICLOPEDIE collection monitored at the Unite de Maladies Génétiques du Globule Rouge [Red Blood Cell Genetic Diseases Unit] at the Centre Hospitalier Universitaire Henri Mondor [Henri Mondor University Hospital Center] in Creteil. This group is composed of adult patients (age≥18 years old) having a hereditary persistence of HbF (HPFH) or intermediate or minor β-thalassemia, and an HbF content of each of their red blood cells which is homogeneous and constant over time. The HbF content of the total red blood cells of patients was determined, in the context of patient monitoring, by regular measurement of the % HbF (by HPLC) and of the mean HbF content per red blood cell (MCHbFCo). Patients having a major sickle cell syndrome (SS, Sβ-Thalassemia or SC) or a sickle cell trait (AS) and also patients treated with an HbF inducer (Hydroxyurea), patients having been transfused during the past 3 months before inclusion and pregnant patients were not included.

In accordance with the Declaration of Helsinki on the ethical principles applicable to medical research involving human beings (World Medical Association, text in force 2008, paragraph 24), all selected patients were informed of the risks and benefits of this study and provided written consent before inclusion. In accordance with the legislation in force (Articles L.1121-3 and R.5121-13 of the French Public Health Code), the information relating to the patients was protected anonymously in order to ensure confidentiality throughout the duration of the study.

This research protocol was approved by the International Review Board Ile-de-France IV located at the Hospital Saint-Louis (IRB No. 00003835).

In summary, 20 ml of blood were taken from the selected patients and collected in 5 tubes containing EDTA. The samples were treated within 24 h after having been taken: determination of the mean HbF concentration (using HPLC), blood hemogram, and flow cytometry analysis then freezing of the remaining samples at −80° C. for the subsequent analyses.

The detailed protocol is explained below.

1. Pre-Treatment of the Samples

20 ml of blood were taken from the selected patients and collected in 5 tubes containing EDTA. The red blood cells were recovered after fractionation of the whole blood by centrifugation at 800 G for 10 minutes at ambient temperature. Approximately 8 ml of blood cell pellet were collected and then washed in 10 ml of phosphate buffer (DPBS 1× Gibco by Life Technologies Cat 14190-094, Life Technologies SAS, Saint Aubin, France) in a 50 ml polypropylene tube. After homogenization, the blood cell suspensions were centrifuged at 1200 G for 5 minutes at ambient temperature. The blood cell pellet was recovered after elimination of the supernatant, and the washing procedure was carried out 3 times.

A first measurement of the HbF content (% HbF by HPLC×MCHCo) was carried out on approximately 1 ml of fresh sample, while 7 ml of blood cell pellet were stored at −80° C. in aliquot fractions of 200 μl in cryotubes (Nalgene Cat 479-6841) using glycerol (B Braun formula No. 569) as cryoprotective agent. For that, approximately 57.7% (V/V) of glycerol were added to the blood cell pellets in 2 steps. Specifically, in order to prevent precipitate formation, ⅓ of the glycerol volume was added dropwise to the blood cell pellet, in a first step, while agitating the cryotube, followed by an incubation for 10 minutes at ambient temperature, and the glycerol volume was subsequently made up with the remaining ⅔.

With this procedure, the red blood cells can be stored for several months, or even years, while at the same time limiting lysis thereof.

The thawing consists of an incubation of the red blood cells in a bath at 37° C. or of rapid agitation in the hand, followed by a series of washing using 2 solutions of NaCl in decreasing concentrations in order to remove the glycerol and to preserve the isotonicity. For that, 75 μl of deglycerolization solution (12% NaCl, B Braun formula No. 570 Melsungen, Germany) were added dropwise in a first step while homogenizing the blood cell suspensions, then the suspensions were incubated at ambient temperature for 10 minutes. Subsequently, 125 μl of NaCl at 0.9% containing glucose were added and the suspensions were incubated for 5 minutes at ambient temperature. Approximately 4×125 μl of NaCl at 0.9% containing glucose were added while observing an incubation of 5 minutes between each volume of NaCl at 0.9% containing glucose. The blood cell suspensions were transferred into 2 ml Eppendorf tubes, then fractionated by gentle centrifugation at 300 G for 10 minutes at ambient temperature while applying a deceleration equal to 5. After removal of the supernatant, 4×125 μl of NaCl at 0.9% containing glucose were added while observing an incubation of 5 minutes between each volume of NaCl at 0.9% containing glucose, then the suspensions were centrifuged as previously. In order to obtain a dry pellet of red blood cells, 500 μl of NaCl at 0.9% containing glucose were added in one step, followed by a centrifugation at 800 G for 10 minutes at ambient temperature. At the end of the final certification, the cell samples can be brought to the desired hematocrit by making up the volume with a buffer solution.

2. Determination of the HbF Content of Each Red Blood Cell

The determination of the HbF content of each red blood cell of the samples from selected patients was carried out both in the fresh cell samples and in the thawed samples using the following 3 methods:

-   -   high-performance liquid chromatography (HPLC) to determine the         mean HbF percentage over all of the red blood cells     -   blood hemogram (including the mean MCHCo); and     -   flow cytometry.

2.1. Determination of the Mean HbF Percentage by HPLC

The determination of the mean HbF percentage by HPLC in the blood cell suspensions was carried out on a Variant II system (Cat: 2702000 Hemoglobin Testing System, Bio-Rad Laboratories, Marnes-la-Coquette, France). It is an ion exclusion liquid chromatography which makes it possible to separate 3 types of hemoglobins: HbF, HbA and HbA2 by virtue of the V2_B-THAL_DU method. The system is composed of a stationary phase composed of resin to which negatively charged carboxyl groups are attached and of a mobile phase composed of two Bis/Tris phosphate buffer solutions having a low and a high ionic strength respectively, and also of a photometric detector emitting 2 different wavelengths: 690 nm and 415 nm for detecting the negative control and the samples respectively. This system makes it possible to quantify the 3 hemoglobins from total blood or from a blood cell suspension with a minimum volume of 500 μl. For a sample of less than 500 μl, a dilution to 1:200 (v/v) with a dilution reagent was carried out before proceeding with the analysis. The analytes were separated as a function of their ionic interaction with the carboxyl groups over a total period of 6 minutes. The HbF was eluted after approximately 0.5 min. The data acquisition was carried out on a CDM 5.2 program in order to determine the mean percentages of each type of Hb. The mean HbF percentage is obtained according to the following relationship: % HbF=(AUC HbF/AUC total Hb)×100 where % HbF represents the mean HbF percentage over all of the erythrocyte cells; AUC HbF represents the area under the curve of the HbF on the chromatogram; AUC total represents the sum of the areas under the curves of the 3 different types of Hb, corresponding in these patients to HbA, HbA₂ and HbF.

2.2. Determination of the Mean Hemoglobin Content Per Red Blood Cell (MCHCo)

The mean hemoglobin content per red blood cell (or mean corpuscular hemoglobin content or MCHCo) constitutes a blood cell constant obtained by complete blood count or blood hemogram. The MCHCo is determined by calculation, by dividing the hemoglobin concentration expressed per liter of blood by the number of red blood cells per liter of blood.

This analysis was carried out on a Horiba ABX Micros ES 60 automated counter (HORIBA Medical, Montpellier, France) on a minimum volume of sample of 500 μl having a hematocrit at approximately 50%.

2.3. Determination of the Mean HbF Content Per Red Blood Cell (MCHbFCo)

The determination of 2 mean parameters (% HbF and MCHCo) obtained by HPLC and by blood hemogram made it possible to calculate the mean HbF content per red blood cell, or MCHbFCo, according to the following relationship: MCHbFCo=(% HbF×MCHCo)/100 where MCHbFCo (pg) represents the mean HbF content per red blood cell; % HbF represents the mean HbF percentage determined by HPLC (see 2.1); MCHCo represents the mean Hb content per red blood cell provided by the automated counter (see 2.2).

3. Determination of the Fluorescence of the Red Blood Cells Labeled with an Anti-HbF Antibody by Flow Cytometry

3.1. Instruments

The analysis of the HbF content of each red blood cell was carried out on an 8-color BD FACS Canto II cytometer (Cat 338960, BD Biosciences, Le Pont de Claix, France) combined with the DIVA® data acquisition software.

Flow cytometry or FACS (fluorescence-activated cell sorting) is a technique which makes it possible to individually analyze the properties (size, granulosity and fluorescence) of particles (beads or cells) in suspension in a flow system.

It is an immunological method based on the immunophenotyping technique which consists of an antigenic detection by virtue of recognition by a specific antibody which is usually conjugated to a fluorescent molecule. The signal measured is principally the fluorescence intensity, which is proportional to the amount of antigens detected by the specific antibody.

The cytometry data acquisition requires pre-processing of the samples, which consists in fixing and permeabilizing the cell membrane, followed by intracellular labeling with an HbF-specific antibody conjugated to a fluorochrome.

3.2. Fixing and Permeabilizing of the Red Blood Cell Membrane

The fixing and permeabilizing of the RBC membrane was carried out using the reagents of a kit for detecting F cells called “Fetal Cell Count™ kit” (Cat IQP-349, IQ Products, Groningen, the Netherlands).

In order to fix the blood cell membrane, 5 μl of washed blood blood cell pellet (fresh or frozen) were added and homogenized in 100 μl of fixing reagent or reagent A containing a preservative or sodium azide (Fetal Cell Count™ kit, Cat IQP-349). 100 μl of buffered formaldehyde solution or fixing solution B (Fetal Cell Count™ kit, Cat IQP-349) were subsequently added to the blood cell suspension. The suspension was homogenized by vortexing and then incubated at ambient temperature for exactly 30 minutes. The blood cell suspension was carefully homogenized every 10 minutes. The RBCs were subsequently washed in 2 ml of 1×PBS buffer containing heparin (reagent D Fetal Cell Count™ kit, Cat IQP-349), followed by centrifugation at 300 G for 3 minutes at ambient temperature. The washed blood cell pellet was resuspended by adding 100 μl of 1×PBS buffer (reagent D Fetal Cell Count™ kit, Cat IQP-349).

In order to permeabilize the blood cell membrane, 100 μl of sodium dodecyl sulfate solution, permeabilization reagent (reagent C Fetal Cell Count™ kit, Cat IQP-349) were added to the suspension of fixed RBCs. The samples were incubated for exactly 3 minutes at ambient temperature. The RBCs were washed as previously. The wash was carried out twice. After removal of the supernatant, the blood cell pellets were resuspended in 1 ml of 1×PBS buffer (reagent D Fetal Cell Count™ kit, Cat IQP-349).

3.3. Intracellular Labeling of the Fixed and Permeabilized Red Blood Cells

An IgG1 Kappa clone mouse monoclonal antibody directed specifically against the gamma chain of human HbF and conjugated with phycoerythrin (PE) (reagent F Fetal Cell Count™ kit, Cat IQP-349, IQ Products) was used to determine the HbF content of each previously fixed and permeabilized red blood cell. For that, 50 μl of antibody solution were deposited in 50 μl of suspension obtained in 3.2. Phycoerythrin, by virtue of its conformational (steric hindrance) properties has the advantage of having a PE:antibody ratio close to 1.

A sample not labeled with the antibody was used as a negative control, while an isotype control was prepared by adding 20 μl of PE-coupled mouse IgG1 Kappa (Cat: 555749, BD Pharmingen™, Le Pont De Claix, France) in 50 μl of suspension obtained in 3.2 in order to verify the binding specificity of the anti-HbF antibody used.

The labeled and nonlabeled samples and also the isotype control were incubated for 15 minutes in the dark and at ambient temperature. The RBCs were subsequently washed in 2 ml of 1×PBS buffer (reagent D Fetal Cell Count™ kit, Cat IQP-349), followed by centrifugation at 300 G for 3 minutes at ambient temperature. The washed blood cell pellets while resuspended by adding 500 μl of 1×PBS buffer (reagent D Fetal Cell Count™ kit, Cat IQP-349). The red blood cells were analyzed in the cytometer within minutes following their pre-processing.

3.4. Cytometer Sample Analysis

During the analysis, the samples were placed in a cold (ice) chamber and in the dark in order to prevent fluorescence losses. Before the data acquisition, the cytometer is precalibrated according to the supplier's recommendations.

The nonlabeled sample was used to determine the voltages (or PMT) of each parameter: FSC (Forward Scatter), SSC (Side Scatter) and fluorescence (table 1). The selection of the red blood cell populations was based on the criteria of size or FSC and granulosity or SSC on the Log FSC-A vs Log SSC-A cytogram while eliminating the debris and background noises. The negative control was determined on the signal generated by the sample not labeled by fluorescence (FIG. 1A). Logarithmic scales were preferred for the FSC and SSC gains.

TABLE 1 Adjustment of the cytometer obtained with the sample of nonlabeled red blood cells Parameter Voltage Log A H W FSC 341 ✓ ✓ ✓ ✓ SSC 328 ✓ ✓ ✓ ✓ PE 400 ✓ ✓ ✓ ✓

The exclusion of the red blood cell (RBC) doublets was carried out with a low flow rate by selecting the RBC populations on the FSC-W vs FSC-H then SSC-W vs FSC-H cytograms according to FIG. 2A and FIG. 2B.

A minimum number of 100 000 RBC events were collected in order to improve the accuracy of the analysis and to register a sufficient number of RBCs after the exclusion of the doublets. For each sample, the statistics were generated by the acquisition software (Diva®) with the number of events recorded for each RBC population selected. The fluorescence intensity (MFI) is given as geometric mean and arithmetic mean.

4. Determination of the Amount of Labeled HbF Antigen Concentration Per Red Blood Cell or AG/RBC

In order to determine the amount of labeled HbF per red blood cell, the fluorescence intensity obtained with the flow cytometer was converted into amount of labeled HbF per red blood cell (AG/RBC) by means of the calibrated beads contained in a phycoerythrin fluorescence quantification kit (Becton Dickinson QuantiBRITE PE, Cat: 340495, BD Biosciences, Le Pont De Claix, France). These beads are conjugated to phycoerythrin in such ways to provide 4 levels of fluorescence (Low, Medlow, MedHigh and High) corresponding to a number of phycoerythrin molecules per bead (FIG. 3A). Before the fluorescence data acquisition in the cytometer, the beads were reconstituted in 500 μl of labeling buffer solution composed of 1×PBS/0.02% NaN₃ (v/v) (Cat: 296028 ChemCruz™ Biochemicals, Santa Cruz Biotechnology, Germany) supplemented with 1% of BSA (Cat: A3803, Sigma, Saint-Quentin Fallavier France). A minimum of 10 000 beads were collected with the same adjustments used for the samples analyzed in point 3.4. The data acquired in the cytometer made it possible to plot a calibration straight line associating Log fluorescence (Log MFI) and Log PE/bead (FIG. 3B). By applying the latter to the fluorescence of the RBCs labeled with the anti-HbF antibody, it is possible to deduce the amount of fluorescent molecules per red blood cell. By using the PE:antibody ratio, the number of fluorescent molecules per red blood cell can be converted into number of antibodies bound per red blood cell, that is to say into number of molecules of antigen (i.e. HbF) per red blood cell (AG/RBC).

5. Determination of the HbF Content (pg) of Each RBC on the Basis of AG/RBC and the MCHbFCo

The data recorded by the acquisition software of the flow cytometer (Diva®) were processed on FlowJo V10 (Cat 130-099-429 Miltenyi Biotec, Paris, France). This is a program for analysis and visualization of the cytometry data exported in “flow cytometry standard” or fcs format. The FlowJo software made it possible to generate the values of each parameter (FSC, SSC, Fluorescence) for each event.

For each RBC sample collected for this study, the distribution of HbF labeled with the antibody is represented by a histogram associating the number of red blood cells and the Log of fluorescence.

The mean HbF content per red blood cell (MCHbFCo) of each sample of selected patients, obtained by the combination of HPLC and of MCHCo, was associated with the mean fluorescence converted into AG/RBC by virtue of the quantification beads.

In order to determine the HbF content (in pg) of each red blood cell, a standard curve was constructed on the basis of the MCHbFCo values and the AG/RBC values, thus giving a range of known MCHbFCo values associated with the fluorescence (figure not presented).

Example 2: Validation of the Method for Determining the HbF Content of Each Red Blood Cell 1. Characteristics of the Included Patients

A cohort of 12 patients was selected in order to constitute a range of HbF content, the criterion for selecting these patients was the confirmation of a homogeneous distribution, Log-Normal of their HbF. The data from these patients were used to construct a standard curve (cf. example 1, point 5) serving to determine the HbF content of each red blood cell. Table 2 summarizes the demographic data and the genetic characteristics of these patients.

TABLE 2 Demographic and biological data of the included patients for constructing the standard curve Age at inclusion Genetic HbF MCHCo MCHbFCo MCV Patients (years) Sex characteristics (%) (pg) (pg) (μm³) 1 18 Male β-thalassemia 100 24.5 24.5 73 2 62 Male β-thalassemia 69.6 27.2 18.93 91 3 50 Male β-thalassemia 5 20.1 12.36 64 4 46 Male β-thalassemia 53.2 19.3 10.27 63 5 50 Female Ghanaian HPFH2 34.80 28 9.74 81 6 39 Female β-thalassemia 27.9 28 7.76 81 7 39 Male β-thalassemia 21 23.8 5 72 8 43 Female β-thalassemia 16.4 23.6 3.87 71 9 37 Male β-thalassemia 14.9 18.5 2.76 62 10 60 Female β-thalassemia 2.7 23.4 3.11 67 11 58 Male β-thalassemia 0.8 19.4 0.15 64 12 23 Male β-thalassemia 0.8 27.8 0.22 83

A homogeneous distribution of the red blood cell fluorescences and therefore of the HbF content was verified on the set of samples collected (FIG. A to FIG. 4N). FIGS. 4C to 4N show, on a semi-logarithmic scale, the Log-normal distribution of the HbF content per RBC of the 12 patients of the range.

2. Test for Reproducibility of the Various Measurements

A reproducibility study was carried out by testing the variabilities on the following measurements:

-   -   MCHbFCo on fresh red blood cells and on red blood cells after a         thawing series     -   MFI values obtained with the QuantiBRITE PE beads and the RBCs     -   AG/RBC after a thawing series

No significant difference was observed on the MCHbFCo values (pg) obtained on fresh red blood cells and after 12 rounds of thawing (n=13, p>0.9999) or on the AG/RBC values measured after 11 rounds of thawing (p=0.73). Likewise, the analysis of variability of the data obtained on the quantification beads revealed no significant difference (n=11, p=0.99).

3. Method for Measuring the HbF Content in Each Red Blood Cell

The MCHbFCo range obtained in the patients having a homogeneous distribution of the HbF contents per red blood cell was associated with the fluorescence intensities and then with the amounts of antigens per red blood cell by means of the calibration beads. A standard curve (or regression straight line) was determined from the AG/RBC means obtained after 11 rounds of thawing and the MCHbFCo values of each patient (FIG. 5).

A coefficient of correlation at 97.18% (r²=0.9444) was obtained between the MCHbFCo values and the means of the amounts of HbF antigen (labeled by the fluorescent antibody) per red blood cell, for the various patients. This standard curve makes it possible to determine the HbF content per RBC for a given fluorescence value. The accuracy of the measurements is given by a confidence interval at 95% determined from the “mean” standard curve, from the standard deviations between each regression and from the number of the regression model having served to calculate the mean.

Example 3: Study of the HbF Distribution During the Treatment with Hydroxyurea 1. Patients

A monocentric, longitudinal, prospective study was carried out on a cohort of 29 adult sickle cell disease patients (age≥18 years old) exhibiting an SS homozygotes mutation, beginning a treatment with hydroxyurea, regularly monitored at the major sickle cell syndrome reference Center at the Centre Hospitalier Universitaire Henri Mondor [Henri Mondor University Hospital Center] in Creteil. Patients having had an attack requiring hospitalization and/or a transfusion exchange program during the previous 3 months before inclusion and also patients who were pregnant or who were wanting to get pregnant were excluded from the study.

The patients exhibiting the eligibility criteria were monitored at various times before and during the treatment with hydroxyurea: D0 (before the beginning of the treatment with hydroxyurea), at 15 days, 1 month, 3 months, 4 months and 6 months or more after the setting up of the treatment with hydroxyurea (D15, M1, M3, M4 and ≥M6 respectively).

In accordance with the Declaration of Helsinki on the ethical principles applicable to medical research involving human beings (World Medical Association, text in force 2008, paragraph 24), all recruited patients were informed of the risks and benefits of this study and provided written consent before inclusion. In accordance with the legislation in force (Articles L.1121-3 and R.5121-13 of the French Public Health Code), the information relating to the patients was protected anonymously in order to ensure confidentiality throughout the duration of the study.

This research protocol was approved by the International Review Board Ile-de-France IV located at the Hospital Saint-Louis (IRB No. 00003835).

2. Methods

During each visit to the center (D0, D15, M1, M3, M4 and ≥M6), approximately 16 ml of blood were taken and collected in 4 tubes containing EDTA.

2.1. Pretreatment of the Blood Cell Samples

The samples were treated within 24 h of being taken. The treatment involves the separation of the red blood cells as a function of their density and the cryopreservation of the blood cell fractions and of the total or non-fractionated red blood cells.

3. Analysis of the Blood Cell Samples

The various measurements carried out on each blood cell fraction or on the total RBCs were composed of:

-   -   complete blood count or blood hemogram by an automated counter;     -   measurement of the mean HbF percentage (% HbF) by HPLC; and     -   flow cytometry measurement of the fluorescence intensity of each         RBC labeled with an antibody directed against HbF in accordance         with the protocol detailed in example 1.

3.1. Complete Blood Count, Measurement of the Mean HbF Percentage (% HbF) by HPLC

These analyses were carried out on fresh RBCs and RBCs after thawing according to the same techniques as those described in example 1.

3.2. Measurement of the HbF Content of Each RBC by Flow Cytometry

This analysis was carried out after thawing of the RBCs stored at −80° C. according to the technique described in example 1.

The HbF content of each RBC was determined using the standard curve described in example 1 based on the fluorescence intensities of each red blood cell, converted into AG/RBC, using the QuantiBRITE PE beads. The measurements were carried out at each stage of the treatment (D0, D15, M1, M3, M4 and ≥M6) in the various blood cell fractions: non-dense, dense and total red blood cells.

4. Statistical Analyses

In the SS patients taking hydroxyurea, the percentages of RBCs containing HbF contents grouped together in arbitrarily defined categories (0 to 2 pg, 2 to 4 pg, 4 to 6 pg, etc.) were compared during the longitudinal monitoring in order to determine the variations in the HbF distribution before and during the treatment with hydroxyurea. The comparisons between each stage of the treatment were carried out by means of an ANOVA measurement repeated between D0, D15-M3 and 2 M6 in the total red blood cells.

The statistical analyses were carried out using the GraphPad Prism®Version 6 software (RITME Informatique Paris France).

5. Results

5.1. Demographic Data and Indications of Hydroxyurea

In total, 29 adult SS patients were included. Out of the entire cohort, 10 patients were monitored between D0 and ≥M6, the RBC samples of a patient were hemolyzed, 9 patients were analyzed.

The demographic data of the included patients are summarized in table 3

TABLE 3 Demographic data of SS patients taking hydroxyurea Men Women N 13 16 Age 33 [28.50; 45] 36 [31; 44.25] Age expressed as Median [25th percentile; 75th percentile]

The hydroxyurea was administered with an average dose of 15 mg/Kg/day for the following indications: VOC prevention, chronic visceral attacks, leg ulcers, sickle cell nephropathy, significant hemolysis with anemia, priapism, stroke

5.2. Biological Data

The mean HbF percentage (% HbF) increases by approximately 3-fold after 6 months of treatment with hydroxyurea. The data are presented in table 4.

TABLE 4 Variation in % HbF during the treatment with hydroxyurea p (D 0 and D 0 D 15-M 1 M 3-M 4 M 6 M 6) Total 2.65 [1.8-6.35] 4.9 [1.85-8.4] 7 [2.4-17.80] 10.05 [3.33-19.13] 0.03 red blood cells Data expressed as Median [25th percentile; 75th percentile]

A significant increase in the mean corpuscular volume (MCV) and in the mean hemoglobin content per red blood cell (MCHCo) was observed after 6 months of treatment with hydroxyurea, indicating good patient conformity and the efficacy of the treatment despite the low patient numbers. The percentage of dense red blood cells has a tendency to decrease; however, this decrease is not significant. Likewise, for the mean corpuscular hemoglobin concentration (MCHC), the leukocytes and the platelets, no significant variation was observed. The results are presented in table 5.

TABLE 5 Biological data before and during the treatment with hydroxyurea D 0 M 6 p Hemoglobin 8 [6.65-9.05] 9.35 [7.30-10.10] 0.2 (g/dl) Leukocytes 10.70 [9.45-12.20] 9 [5.35-11.58] 0.11 (Giga/l) MCV 87 [83-95] 100.50 [87.75-115] 0.03 (femtoliter) MCHC (g/dl) 32.60 [32.05-35.10] 34.40 [32.43-35.70] 0.08 MCHCo (pg) 29.70 [25.30-32.85] 33.80 [30.70-39.65] 0.008 Platelets 359 [288-548] 318 [257-438.80] 0.18 (giga/l) Data expressed as Median [25th percentile; 75th percentile]

5.3. HbF Distribution During the Treatment with Hydroxyurea

The qualitative analysis by flow cytometry of the response to treatment with hydroxyurea shows a heterogeneous HbF distribution before the beginning of the treatment in 8 patients. After 6 months of treatment with hydroxyurea, the HbF follows a homogeneous distribution in 6 patients. The HbF distribution becomes homogeneous starting from M2 in 1 patient and after ≥M4 in 5 patients. FIGS. 6A to 6K illustrate the variations in HbF distribution during the treatment with hydroxyurea in the total red blood cells.

The HbF distribution was quantitatively analyzed by means of the method described in study 1. The number of red blood cells per HbF content range (pg) was determined for each patient. The analysis of the red blood cell distribution over time while taking hydroxyurea was carried out in 9 patients. The results show a significant variation in the red blood cells having the lowest HbF contents (0 and 2 pg) with an increase of approximately 25% between D0 and M6. The red blood cells which have an HbF content of greater than 20 g increase significantly, by up to 3 times, after 6 months of treatment with hydroxyurea; however, this variation is not significant. The red blood cells which have medium-high contents exhibit much lower variations.

TABLE 6 Red blood cell distribution per HbF content range under hydroxyurea treatment D 0 D 15-M 3 ≥M 6 HbF (pg) (% RBCs) (% RBCs) (% RBCs) p [0; 2[ 71.13 (23.16 65.63 (25.13 53.17 (30.63 0.0083 [2; 4[ 9.14 (7.44 11.32 (6.80 15.05 (10.11 0.1868 [4; 6[ 4.54 (3.86 4.88 (2.90 6.70 (4.39 0.2226 [6; 8[ 3.16 (2.92 3.42 (2.52 3.92 (2.44 0.5186 [8; 10[ 2.42 (2.32 2.65 (2.18 2.72 (1.83 0.6932 [10; 15[ 1.94 (1.84 2.41 (2.73 2.55 (2.57 0.3926 [15; 20[ 3.66 (3.35 4.42 (4.27 4.29 (3.42 0.474 ≥20 4.01 (4.35 5.27 (8.56 11.60 (18.64 0.1626 The data are expressed as mean (standard deviation)

The measurement of the fluorescence intensities of each red blood cell by flow cytometry made it possible to categorize each red blood cell as a function of the HbF content thereof. The analysis of the red blood cell distribution as a function of the HbF content thereof makes it possible to identify various treatment response profiles. By way of example, patients 11 and 24 (tables 7 and 8), expressing identical % HbF values at D0 (6%) and M6 (18%) with an MCHCo at D0=33.6 and 34.5 pg and at M6=38.9 and 42 pg (patients 11 and 24 respectively), exhibit 2 different distributions. In patient 11, a lower number of red blood cells is noted with an HbF content of less than 2 pg with a decrease of approximately 73% at M6, whereas patient 24 exhibits a higher number of red blood cells with an HbF content of less than 2 pg with a decrease of approximately 40% at M6. The HbF distribution in patient 11 is described by a bimodal curve at D0 and at M6, whereas the distribution of patient 24 exhibits a relatively homogeneous distribution over the low HbF amounts with a clear shift of the set toward higher amounts.

Patient 11

D 0 D 15-M 1 M 3-M 4 ≥M 6 HbF (pg) (% RBCs) (% RBCs) (% RBCs) (% RBCs) [0; 2[ 48.44 55.38 48.83 12.93 [2; 4[ 26.27 23.31 31.73 17.62 [4; 6[ 8.13 6.25 9.30 11.85 [6; 8[ 3.77 3.07 4.32 8.05 [8; 10[ 2.69 2.55 2.04 6.08 [10; 15[ 2.41 2.24 0.72 6.13 [15; 20[ 4.21 4.42 2.46 9.77 ≥20 4.08 2.78 0.59 27.57 Patient 24

D 0 D 15-M 1 M 3-M 4 ≥M 6 HbF (pg) (% RBCs) (% RBCs) (% RBCs) (% RBCs) [0; 2[ 60.82 53.17 44.56 36.98 [2; 4[ 9.38 11.57 24.93 25.28 [4; 6[ 4.83 6.69 12.53 13.64 [6; 8[ 4.01 5.58 5.81 6.92 [8; 10[ 3.63 4.50 3.09 3.93 [10; 15[ 3.83 3.89 1.87 2.70 [15; 20[ 6.38 7.56 3.81 5.36 ≥20 7.12 7.04 3.41 5.20

Tables 7 and 8: distribution of each red blood cell as a function of the content thereof of HbF categories (compared with total red blood cells) before and during the treatment with hydroxyurea.

The biological data of patients 11 and 24 were also analyzed (table 9 below).

TABLE 9 Biological data of patients 11 and 24. Relative Relative difference in difference in Patient 11 Patient 11 Patient 24 Patient 24 % D 0 vs M 6 % D 0 vs M 6 (D 0) (M 6) (D 0) (M 6) (patient 11) (patient 24) HbF (g/dl) 6.2 7.1 7.6 8.7 +14.5 +14.5 RETIC (g/l 275 63 271 116 −77.1 −57.2 LDH (IU/l) 905 383 509 303 −57.7 −40.5 ASAT (IU/l) 55 26 32 29 −52.7 −9.4 HbF = Hemoglobin F; RETIC = Reticulocytes; LDH = Lactate dehydrogenase; ASAT = Aspartate transaminase

The biological data showed that patients 11 and 24 exhibit identical relative differences in HbF (+14.5%) before and after six months of treatment with hydroxyurea. They nevertheless exhibit considerable differences in terms of HbF distribution (see table 8). Patient 11, compared with patient 24, exhibits, before treatment (D0), fewer RBCs having high protective HbF contents (17% RBCs vs 25% RBCs>6 pg of HbF), which might explain a greater anemia (6.2 g/dl vs 7.6 g/dl) and a greater hemolysis (LDH at 905 IU/I vs 509 IU/I). On the other hand, the increase in RBCs>6 pg HbF in patient 11 was more marked than in patient 24, at 6 months of treatment (57% vs 24%), and might explain a greater decrease in hemolysis under treatment (RETIC: −77% vs −57%; LDH: −57% vs −40.5%; ASAT −52% vs −9.4%).

Another example of distribution as a function of the HbF content of each red blood cell use represented for patient 2. Patient 2 exhibited, before the treatment with hydroxyurea, a relative homogeneity of HbF content distribution. However, at M6, the distribution was characterized by a clear increase in red blood cells containing a content greater than 20 pg to the detriment of all the other categories (table 10). It should be noted that the % HbF varies from 6% to 25% between D0 and M6, which is similar to the 2 previous examples.

TABLE 10 distribution of red blood cells as a function of the content thereof of HbF categories (compared with total red blood cells) before and during the treatment with hydroxyurea for patient 2 D 0 D 15-M 1 M 3-M 4 ≥M 6 HbF (pg) (% RBCs) (% RBCs) (% RBCs) (% RBCs) [0; 2[ 22.43 9.21 5.29 4.98 [2; 4[ 15.76 14.72 8.62 7.04 [4; 6[ 12.85 9.52 10.61 5.53 [6; 8[ 10.04 8.47 10.39 4.85 [8; 10[ 7.84 7.35 8.75 4.56 [10; 15[ 5.79 9.03 8.50 7.48 [15; 20[ 11.05 14.27 14.54 9.75 ≥20 14.24 27.43 33.30 55.81

6. Conclusion

The data showed that the red blood cells having a very low HbF content decreased greatly and that the red blood cells having an HbF content greater than 20 pg increase considerably, up to 3-fold, after 6 months of treatment with hydroxyurea. 

The invention claimed is:
 1. A method for determining, in vitro, a hemoglobin F (HbF) content of each erythroid cell of a set of erythroid cells contained in a sample of erythroid cells, comprising the steps of: a) isolating erythroid cells from the sample; b) permeabilizing the membrane of the isolated erythroid cells; c) labeling the HbF of the erythroid cells obtained in step b) with an anti-HbF antibody conjugated to a fluorochrome capable of emitting fluorescence; d) measuring, by flow cytometry, fluorescence intensity (MFI) of each erythroid cell of the set of erythroid cells; and e) determining the hemoglobin F content of each erythroid cell of the set of erythroid cells by comparing the fluorescence intensity of each erythroid cell with a standard curve associating the fluorescence intensity measured for an erythroid cell with the HbF content.
 2. The method as claimed in claim 1, wherein the membrane of the isolated erythroid cells is fixed before the permeabilization step.
 3. The method as claimed in claim 2, wherein the membrane of the isolated erythroid cells is fixed with sodium azide and/or formaldehyde.
 4. The method as claimed in claim 1, wherein said sample is a blood sample.
 5. The method as claimed in claim 4, wherein the blood sample is a human blood sample.
 6. The method as claimed in claim 1, wherein said fluorochrome is phycoerythrin (PE).
 7. The method as claimed in claim 1, wherein said anti-HbF antibody is directed against the gamma chain of HbF.
 8. The method as claimed in claim 1, wherein the membrane of the isolated erythroid cells is permeabilized with sodium dodecyl sulfate.
 9. The method as claimed in claim 1, wherein the hemoglobin F content is determined for each erythroid cell of a set of at least 10,000 erythroid cells.
 10. The method as claimed in claim 9, wherein the hemoglobin F content is determined for each erythroid cell of a set of at least 100,000 erythroid cells.
 11. The method as claimed in claim 9, wherein the hemoglobin F content is determined for each erythroid cell of a set of between 50,000 and 100,000 erythroid cells.
 12. The method as claimed in claim 1, wherein the erythroid cells are red blood cells.
 13. A method for determining, in vitro, an amount of red blood cells transfused into a patient suffering from sickle cell disease or β-thalassemia, comprising: a) determining the hemoglobin F (HbF) content of each red blood cell of a set of red blood cells of a sample of red blood cells from the patient according to the method of claim 12; b) using the hemoglobin F (HbF) content of each red blood cell determined in step a) to determine the amount of red blood cells transfused into the patient, said transfused red blood cells having an HbF content substantially equal to zero (=0 pg).
 14. An in vitro method for monitoring a therapeutic efficacy of a treatment for sickle cell disease or for β-thalassemia, comprising: a) determining the hemoglobin F (HbF) content of each red blood cell of a set of red blood cells of a sample of red blood cells from a patient having undergone a treatment for sickle cell disease or for β-thalassemia according to the method of claim 12; b) using the hemoglobin F (HbF) content of each red blood cell determined in step a) in the monitoring of the therapeutic efficacy of the treatment for sickle cell disease or for β-thalassemia, in which a therapeutic efficacy is observed when at least a predetermined percentage of red blood cells of the set of red blood cells has an HbF content which exceeds a reference threshold and/or when a decrease in amount of red blood cells having an HbF content <2 pg is observed.
 15. The method as claimed in claim 14, wherein the reference threshold is 2 pg or more.
 16. The method as claimed in claim 14, wherein the reference threshold is selected from the group consisting of: 3 pg, 4 pg, 5 pg, 6 pg, 7 pg, and 7.5 pg. 